Chromosome-level genome assembly of Plazaster borealis sheds light on the morphogenesis of multiarmed starfish and its regenerative capacity

Abstract Background Plazaster borealis has a unique morphology, displaying multiple arms with a clear distinction between disk and arms, rather than displaying pentaradial symmetry, a remarkable characteristic of echinoderms. Herein we report the first chromosome-level reference genome of P. borealis and an essential tool to further investigate the basis of the divergent morphology. Findings In total, 57.76 Gb of a long read and 70.83 Gb of short-read data were generated to assemble a de novo 561-Mb reference genome of P. borealis, and Hi-C sequencing data (57.47 Gb) were used for scaffolding into 22 chromosomal scaffolds comprising 92.38% of the genome. The genome completeness estimated by BUSCO was 98.0% using the metazoan set, indicating a high-quality assembly. Through the comparative genome analysis, we identified evolutionary accelerated genes known to be involved in morphogenesis and regeneration, suggesting their potential role in shaping body pattern and capacity of regeneration. Conclusion This first chromosome-level genome assembly of P. borealis provides fundamental insights into echinoderm biology, as well as the genomic mechanism underlying its unique morphology and regeneration.

We revised all the suggested sentences in the manuscript. Echinoderms are marine animals characterized by the following three remarkable 45 characteristics: 1) extensive regenerative abilities in both adult and larval forms [1,2], 2) the 46 water vascular system used for gas, nutrient and waste exchange [3], and 3) extraordinary morphological characteristics including pentaradial symmetry [4,5].
Pentaradial symmetry has been observed in all extant classes of echinoderm. Echinoids (sea 49 urchin) and holothurians (sea cucumber) always have five ambulacral grooves, and crinoids 50 have many arms in multiples of five that branch out from the five primary brachia [4,5]. Most 51 species of asteroids and ophiuroids are five-armed, but many exceptions are scattered across 52 the tree of Echinodermata. Extant asteroids are distinguished by 34 families, including 20 53 families of only five-armed species, nine families of both five-armed and multi-armed species, 54 and five families with exclusively multi-armed species [6]. However, most multi-armed forms 55 have arm numbers that cannot be divided into five, raising questions about the arm 56 development mechanisms that do not follow the pentaradial symmetry.  successfully annotated with at least one related functional assignment (Table 3).   conservation of synteny with P. ochraceus than A. rubens, which seems to be influenced by the 139 observed genomic region. We also analyzed synteny of P. borealis with A. planci, the starfish 140 of a different order; however, chromosomes were not matched. These results suggest that 141 genomes within the Forcipulatida order are remarkably conserved in terms of synteny, allowing 142 us to confirm the high quality of our genome assembly.    Table 3). 176 In addition, we identified 607 gene families unique in P. borealis consisting of 2,631 genes and 177 111 one-to-one orthologous genes between P. borealis and six other species. The gene families  Table 5). It is worth noting that these positively selected genes also included BMP4, which 212 regulates regeneration and tissue specification (Table 4).

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Regeneration is a high-energy-required process in which starfishes in the regeneration state 214 increase the amount of lipid and energy in the pyloric caeca to use [30]. GPR161 and BMP4, 215 well-known genes to be critical in regeneration, were also detected as positively selected genes.

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The G-protein coupled receptor Gpr161 negatively regulates the Hedgehog pathway via cAMP 217 signaling, known to participate in the process of tissue regeneration [31,32]. Additionally, 218 previous studies of planarian regeneration indicate that BMP4 is a key for tissue specification, 219 especially dorsal-ventral polarity, which may explain the distinctive disk of P. borealis [33].

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Together with those of previous studies, our results further suggest that related genes may have 221 contributed to the regeneration and development of the unique body plan of P. borealis, multiple arms. Therefore, P. borealis can be potentially regarded as a valuable model to 223 investigate the mechanisms underlying supernumerary arm development and regeneration.

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This high-quality genome is useful and valuable genetic resource for future research, especially 225 in a unique body plan and regeneration biology.   We also used an Illumina platform to generate short high-quality sequencing reads. DNA

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We also used an Illumina platform to generate short high-quality sequencing reads. Using  To assemble transcriptome, we selected hybrid approach to restore more known genes and 339 discover alternatively spliced isoforms, which can be useful in transcriptome analysis of previously unsequenced organism. Therefore, long reads and short reads from three tissues 341 were used for assembly. To ensure the accuracy of subsequent analyses, we trimmed the raw 342 reads to remove adaptor sequences and low-quality reads. Trimmomatic v0.39 (Trimmomatic,